405 Central European Journal of Urology

Original Paper UROLOGICAL ONCOLOGY

MMP7 and MMP8 genetic polymorphisms in bladder cancer patients Edyta Wieczorek1, Edyta Reszka1, Wojciech Wasowicz1, Adam Grzegorczyk2, Tomasz Konecki2, Marek Sosnowski2, Zbigniew Jablonowski2 1Department of Toxicology and Carcinogenesis, Nofer Institute of Occupational Medicine, Łódź, Poland 21st Department of Urology, Medical University, Łódź, Poland

Article history Introduction. Breakdown of the by matrix (MMPs), as we know, Submitted: Jun. 16, 2013 is one of mechanisms involved and required in tumor invasion. MMP7 is a negative prognostic factor Accepted: Oct. 24, 2013 of various malignances, while MMP8 exhibits an inhibitory effect on tumorigenesis and metastasis. We evaluated the potential association of functional polymorphisms in the promoter of the MMP7 (rs11568818) and MMP8 (rs11225395) and bladder cancer (BCa) risk.

Materials and methods. The study included 241 BCa cases and 199 healthy population controls that were collected at the First Department of Urology, Medical University (Łódź, Poland) and at the Nofer Institute of Occupational Medicine (Łódź, Poland). Genomic DNA samples were isolated from venous blood and genetic polymorphisms were analyzed by real–time polymerase chain reaction using TaqMan fluorescent probes. Associations between genotype and allele status were estimated by logistic regression models adjusted for classic risk factors (e.g. age, gender and cigarette smoking). Results. MMP7 and MMP8 genotypes were distributed similarly in BCa patients and in controls and at Correspondence Edyta Wieczorek least one variant allele was not associated with BCa cancer risk (OR, 0.91; 95% CI, 0.60–1.39; p = 0.662 Nofer Institute of for MMP7 and OR, 0.96; 95% CI, 0.63–1.46; p = 0.836 for MMP8). We observed higher prevalence of Occupational Medicine MMP7 GG genotypes among BCa patients than in controls (OR, 1.54; 95% CI, 0.93–2.55;p = 0.093). 8, Św. Teresy od Dzieciątka Additionally, genetic polymorphisms in theMMP7 and MMP8 were not associated with the tumor grade Jezus Street 91–348 Łódź, Poland or stage. phone: +48 42 631 46 25 Conclusions. Our results suggest that genetic variations in two genes encoding members of theMMP7 [email protected] and MMP8 are not associated with a risk of BCa in the Caucasian population.

Key Words: MMP ‹› genetic polymorphism ‹› case–control study ‹› bladder cancer

INTRODUCTION currences of MIBC and are related with an increased risk of metastases and low survival [4]. Therefore, Bladder cancer (BCa) was estimated to be the sev- more extensive studies are currently in progress to enth most common type of cancer worldwide in 2009 elucidate the importance of the various tumor char- [1]. In Poland, BCa was the third most frequent ma- acteristics or genetic factors in the areas of BCa risk, lignant tumor in men and thirteenth in women in recurrence or progression [5, 6]. 2009 [2]. Etiology of BCa includes environmental fac- MMPs are implicated in various stages of physiologi- tors such as exposure to tobacco smoke, occupational cal processes such as embryonic development, ovu- exposure, and geography of selected infectious dis- lation, wound healing, and pathophysiological pro- eases like schistosomiasis [3]. The group of patients cesses like heart failure, arthritis, atherosclerosis, with non–muscle–invasive disease (NMIBC) covers periodontal disease, bone remodeling, and carcino- 75% and muscle invasive disease (MIBC) covers 25% genesis. MMPs can hydrolyze most of the extracel- of BCa patients. About 20% of NMIBC occur as re- lular matrix (ECM) components, a range of non–

Cent Eur J Urol 2013; 66: 405-410 20.4524/ceju.2013.04.art3 406 Central European Journal of Urology matrix , matrix substrates, and bioactive es between MMP7 or MMP8 genotypes and tumor substrates. MMPs can also release from the ECM grade or stage, patients with BCa were divided into the signaling molecules important in tumor progres- categories: group 1) with G1, and group 2) with G2 sion and metastasis [7]. MMPs are the that and G3 or group 1) with T1, and group 2) with T2– are able to organize the tissue architecture required T4, respectively. All patients with missing data in the process of carcinogenesis and contribute to cell were excluded from these analyses. Additionally, to adhesion, epithelial to mesenchymal transition, and examine the joint effects of MMP7 or MMP8 geno- tumor angiogenesis [8]. types and tobacco smoking status on BCa risk, we MMP7 also known as matrilysin–1 or PUMP–1, pos- stratified cases and controls into three categories: sesses broad substrate specificity, and can degrade 1) never smokers – persons who had never smoked types: IV, V, IX, X, XI, aggrecan, entactin, in their lifetime, 2) ex–smokers – individuals who laminin, vitronectin, fibrin/fibrinogen, tenascin, gel- were abstinent for at least 1 year before the inter- atin, fibronectin, and other substrates of the ECM view, 3) smokers – persons who stated they current- components [7]. MMP7 expression can promote ly smoked cigarettes or who were abstinent for up cancer cell growth and thus can be associated with to 1 year before the interview. The study was ap- tumor invasion, recurrence and poorer survival proved by the Local Ethical Committee of the Nofer for various malignances [9–16]. MMP8, commonly Institute of Occupational Medicine. known as –2 or , hydrolyzes collagen types: I, II, III, VII, X, gelatin, DNA isolation and genotyping entactin, tenascin, aggrecan, and other ECM compo- nents [7]. MMP8 is one of MMPs that has been found The genomic DNA was isolated from the peripheral to produce anticancer effects due to its ability to in- blood samples of the study subjects using commer- hibit tumorigenesis and metastasis [17, 18]. Cancer cial DNA kits QIAamp DNA Mini Kits (Qiagen) fol- studies conducted over eight years have found an as- lowing the manufacturer’s protocol. The promoter sociation between the development of specific cancer single nucleotide polymorphisms (SNPs) in the sites and functional polymorphisms that can alter MMP7 (rs11568818) and MMP8 (rs11225395) genes expression located in the promoter regions of were genotyped using TaqMan fluorescent probes, MMP7 (rs11568818) and MMP8 (rs11225395) [19– assays on demand (Assay ID: C_27852953_10 and 23]. However, there have been no association studies C_1366493_10, respectively) (Life Technologies). All of MMP7 and MMP8 polymorphisms in BCa risk in patients and controls were genotyped using Real– the Caucasian population. Therefore, the objective Time PCR CFX96 System (BioRad). of this association study was to evaluate polymor- phisms in MMP7 and MMP8 and characterize their Statistical analysis associations with BCa susceptibility in a population of patients from Łódź. Group characteristics were determined by the χ2 (chi–square) test and Student’s t–test. Hardy–Wein- MATERIALS AND METHODS berg equilibrium (HWE) was tested by comparing the observed and expected genotype frequencies in Study population cases and controls at equilibrium based on the χ2 test at a significant level of p <0.05. The strength of the BCa patients were recruited from the First Depart- association between genetic polymorphisms in the ment of Urology, Medical University of Łódź and MMP and BCa susceptibility was measured by odds Nofer Institute of Occupational Medicine in Łódź ratios (ORs) corresponding to 95% confidence inter- from 2007 to 2013. The 241 BCa patients and 199 val (95% CI). ORs and 95% CI were determined by lo- healthy population controls were recruited from an gistic regression analyses using additive models that ethnically homogeneous Polish population. Data included adjustment for age, gender, and cigarette on histological cancer grades diagnosed at the smoking status. Major allele homozygotes served as First Department of Urology were not accessible the reference group, and heterozygotes and minor for all cases. All of the BCa patients underwent allele homozygotes were separately compared. The transurethral resection and had histopathologi- association between the MMPs genotype frequencies cally confirmed NMIBC or MIBC at various tumor and characteristics was estimated by Fisher’s exact (T) stage and grade (G) of neoplasm. The tumor test using the Stata 11 (StataCorp LP, USA) soft- stage (87.1% of diagnosed BCa patients), and tumor ware. All statistical tests presented in this paper are grade (89.2% of diagnosed patients) were included two–sided and p values were considered to be statis- in the association analyses. To evaluate differenc- tically significant when p ≤0.05. 407 Central European Journal of Urology

RESULTS Table 2. Summary of the genetic polymorphisms in the MMP analyzed in this study Cohort characteristics Gene MMP7 MMP8 We analyzed genetic polymorphisms in the promoter MMPs exert cancer effects Supporting Inhibiting region of MMP7 and MMP8 in 241 BCa patients and dbSNP ID rs11568818 rs11225395 199 controls from Poland. In Table 1 we summarized Nucleotide positiona –181 –799 demographics, cigarette smoking status, and clinical Type of region Promoter Promoter characteristics of the patients. Statistically signifi- 11q22.2a 11q22.2b cant differences were observed between BCa cases b and controls in terms of the distribution of gender RefSNP Alleles A/G C/T Allele with greater transcriptional (p = 0.001) and tobacco smoking status (p = 0.001). G allele T allele At the time of BCa diagnosis, patients were between activity ages 64.9 and 67.7 years (mean 66.1 ±10.4) and were A 0.55 0.56 at the same age as the controls (mean 66.3 ±10.6) (p Alleles frequency of BCa patientsc B 0.45 0.44 = 0.872). The genotype distributions of both chosen HWEd 1.06 0.64 polymorphisms were consistent with Hardy–Wein- A 0.58 0.56 berg equilibrium (Table 2). Alleles frequency of controlsc B 0.42 0.44 Analysis of association HWEd 1.01 0.15 aminus indicates promoter region; bmajor/minor alleles and frequencies, as For the genetic polymorphisms in the MMP7 determined by the distribution among the case and controls; cA major allele, (rs11568818) and MMP8 (rs11225395) genes, sig- B minor allele; dHardy–Weinberg equilibrium p–value nificant differences between genotyped BCa patients and controls were not observed. Prevalence of MMP7 G/G genotype was only slightly different among pa- tients (22.1%) and controls (16.1%), and BCa risk Table 1. Selected characteristics of the BCa patients and among MMP7 G/G was estimated to OR, 1.54; 95% healthy controls at the time of diagnosis CI, 0.93–2.55; p = 0.093 (Table 3). Association of Variable Controls n (%) Cases n (%) p–valueb single MMP7 and MMP8 genotypes with BCa risk in groups stratified by tobacco smoking status was Gender assessed. The statistical significance of additive ef- Female 23 (11.6) 59 (24.5) Male 176 (88.4) 182 (75.5) 0.001 fects was not observed among stratified groups (data not shown). We also studied MMP7 and MMP8 poly- Mean age (years) ±SD 66.1 ±10.4 66.3 ±10.6 0.872c morphisms and their risk associated with different a Smoking status histology grade of BCa. None of the G and T–cate- d Never–smokers 70 (35.4) 64 (27.0) gorized groups of patients were associated with in- Ex–smokerse 91 (45.9) 92 (38.8) dividual MMP7 or MMP8 polymorphisms (data not Smokersf 32 (18.7) 81 (34.2) 0.001 shown). In order to examine whether the effect of a Tumor grade gene–gene interactions in MMP7 and MMP8 poly- G1 – 113 (46.9) morphisms might change the BCa risk, we analyzed G2 – 64 (26.6) G3 – 38 (15.8) various combinations of genotypes. We observed that G2 and G3 – 102 (42.3) the combined effect of MMP7 and MMP8 genotypes Unknown – 26 (10.7) – did not provide statistically significant differences Tumor stagea between cases and controls (Table 4), nor in any to- NMIBC (T1) – 169 (70.1) bacco smoking status (data not shown). MIBC (T2–T4) – 41 (17.0) Unknown – 31 (12.9) – DISCUSSION a the number of cases and controls may vary because of some missing It is generally believed that the development of the data; bchi–square test was used to determine p–value; ct–test was used to determine p–value; d never smokers – persons who had never smoked; invasive phenotype (advanced metastatic stage of eex–smokers – individuals who were abstinent for at least 1 year;f smokers cancer) and further poor prognosis may be associ- – individuals who currently smoke or who were abstinent for up to 1 years ated with increased expression of MMPs [22, 24, before the interview; NMIBC – non–muscle–invasive bladder cancer; MIBC– 25]. Furthermore, we know that MMPs regulatory muscle–invasive bladder cancer effect may be related to MMP promoter SNPs. It is 408 Central European Journal of Urology

Table 3. Distribution of the genetic polymorphisms in the MMP7 and MMP8 among controls and BCa patients

Controlsb Casesb BCa risk OR (95% CI)c, p–value Gene dbSNP Genotypesa n (%) n (%) Crude Adjustedd A/A 63 (31.6) 76 (31.7) Ref. Ref. MMP7 A/G 104 (52.3) 111 (46.3) 0.79 (0.54–1.14), 0.210 0.72 (0.49–1.08), 0.112 (rs11568818) G/G 32 (16.1) 53 (22.1) 1.48 (0.91–2.40), 0.114 1.54 (0.93–2.55), 0.093 A/G+G/G 136 (68.3) 164 (68.3) 1.00 (0.67–1.50), 0.999 0.91 (0.60–1.39), 0.662 C/C 60 (30.2) 72 (29.9) Ref. Ref. MMP8 C/T 101 (50.8) 125 (51.9) 1.04 (0.72–1.52), 0.816 0.97 (0.65–1.44), 0.880 (rs11225395) T/T 38 (19.1) 44 (18.2) 0.94 (0.58–1.53), 0.822 1.00 (0.60–1.66), 0.997 C/T+T/T 139 (69.9) 169 (70.1) 1.01 (0.67–1.53), 0.950 0.96 (0.63–1.46), 0.836 agenotypes and frequencies, as determined by the distribution among the cases and controls;b the number of cases and control may vary because of some missing data; cOR, odds ratio, CI, 95% confidence interval;d odds ratio adjusted for age, gender and cigarette smoking status

Table 4. Risk of BCa associated with the gene-gene gated serum and plasma levels of MMP7, identified interactions in MMP7 and MMP8 polymorphisms high levels of MMP7 as predictors of BCa outcomes. Moreover, MMP7 expression can correlate Genotype Genotype Controls Cases OR (95% CI)a, with pathologic parameters like tumor stage, tumor MMP7 MMP8 n (%) n (%) p–value Crude grade, and metastasis in BCa [29, 30, 31]. C/C 16 (25.4) 15 (19.7) Ref.b Recent studies on metastatic potential of breast can- A/A C/T+T/T 47 (74.6) 61 (80.3) 1.38 (0.62–3.08), 0.540 cer cells show that over–expression of MMP8 protein C/C 44 (32.4) 57 (34.8) 1.38 (0.62–3.10), 0.537 provides a protective effect in the metastatic process A/G+G/G [32]. Also, in studies of the squamous cell carcinoma C/T +T/T 92 (67.6) 107 (65.2) 1.24 (0.58–2.65), 0.699 of the tongue it has been found that MMP8 protein aOR, odds ratio, CI, 95% confidence interval;b major allele homozygotes expression is correlated with improved survival of pa- (reference group) tients [34]. However, there are only a few case–con- trol studies regarding MMP8 –799 C/T (rs11225395) impact on cancer development. In vitro data has sug- considered that the analysis of SNPs in MMPs as gested that promoter MMP8 polymorphism has pu- prognostic biomarkers, might identify BCa patients tative functional significance and higher transcrip- at an increased risk of lymph node metastasis even tion activity for minor MMP8 T allele compared with before surgery. MMP gene variants may influence MMP8 C allele [35]. Indeed, breast cancer risk (OR, their transcriptional activity and protein expression, 0.7; 95% CI, 0.5–0.9) and survival (OR, 0.4; 95% CI, function, and activity. Therefore, they may be asso- 0.2–0.8) was significantly reduced for women carry- ciated with cancer risk, prognosis, and responses to ing the MMP8 allele T [35, 36]. However, a study of treatment. Polish individuals reported that allele T was asso- MMP7 and MMP8 may play different roles in can- ciated with risk of developing malignant melanoma cer risk and cancer progression. MMP7 over–expres- [37]. In analyzed individuals from Central Poland, sion is often observed in various malignancies, which the frequency of MMP7 G alleles was 0.42, while for supports action on carcinogenesis, while MMP8 pos- MMP8 T it was 0.44. To compare, in control indi- sesses inhibitory effect [16, 17, 26]. Genetic polymor- viduals from Łódź, the frequency of MMP7 G alleles phism in the MMP7 –181 A/G (rs11568818) promoter was 0.45 [38], while in individuals from Szczecin, the have been described in several association studies on frequency was 0.39 [37]. lung, breast, colorectal, gastric, and prostate cancer, In the present association study we tested the hypoth- indicating significant association between at least esis whether the functional genetic polymorphisms one MMP7 G allele and gastric cancer risk (OR, 1.90; in the MMP7 and MMP8 genes contributed to BCa 95% CI, 1.43–2.51), but no consistent results for oth- risk. To our knowledge, this is the first study investi- er types of cancer [27]. MMP7 genetic polymorphism gating the relationship between MMP7 (rs11568818) affects the transcriptional activity of that gene and and MMP8 (rs11225395) polymorphisms and BCa leads to changes in its expression. Namely, the G al- risk in Caucasian population. Two studies con- lele compared to the A allele is transcriptionally more ducted by Srivastava et al. in North India popula- active [28]. Expression of MMP7 has been detected tion showed significantly higher risk of BCa among in various cancers [16, 26]. Studies which investi- MMP7 G/G (rs11568818) individuals (OR, 2.38; 95% 409 Central European Journal of Urology

CI, 1.30–4.34), while MMP8 C/T+T/T (rs11225395) evant candidate genes such as MMP7 (rs11568818) genotype carriers possessed reduced BCa risk (OR, and MMP8 (rs11225395) and BCa risk in the Cau- 0.27; 95% CI, 0.10–0.69) [19, 23]. Nevertheless, we casian population. It should be noted that our study found that these genetic polymorphisms do not con- was encumbered with a limitation. Due to the small tribute to risk of BCa and cancer grade. However, we number of cases studied, some associations were not observed that individuals with MMP7 G/G genotype evident. The search for new biomarkers of cancer is were at statistically borderline risk of BCa (OR, 1.54; still required and more extensive research on genetic 95% CI, 0.93–2.55; p = 0.093). We found no additive polymorphisms of MMP in BCa should be undertak- effect of smoking and genetic polymorphisms in the en in the future. MMP7 and MMP8 among never–smokers, ex–smok- ers, and smokers. Additionally, our investigation of the potential combined effect of gene–gene interac- tion between genetic polymorphisms, reported no correlation with BCa risk and cancer grade.

CONCLUSIONS ACKNOWLEDGEMENTS This work was supported by Polish Society of Urology, Internal In this study, the results show no association be- Grant IMP 1.22 and the Ministry of Science and Higher Education tween the genetic polymorphisms in biologically rel- (2012/05/B/NZ5/01406).

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